Plasma protein receptor purification.
klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Fri Aug 17 08:12:26 EST 2001
s010867 at mailserv.cuhk.edu.hk (Daniel) wrote:
>I wanna isolate receptor protein from culture cell. The theoretical
>pI/Mw of that protein is 8.53/78KDa. I've tried several methods but I
>couldn't get any postive signals from my western blotting. I think it
>maybe the result of low abundance of that protein on these cell. Can
>anybody give me some suggestion on the receipe of buffer and better
>methods for me to isolate these protein?
First, you must find a detergent that keeps your receptor active
after membrane solubilization. Second, you must have a working
deytection system (Western or ligand binding).
Given that, two generic purification steps are obvious:
affinity chromatogrphy on immobilized ligand and cation exchange
(S- or CM-based, whichever works best) at ~ neutral pH.
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